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This Provisional PDF corresponds to the article as it appeared upon acceptance. Fully formatted PDF and full text (HTML) versions will be made available soon. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro Journal of Neuroinflammation 2011, 8:183 doi:10.1186/1742-2094-8-183 Francesco Boato (fraboato@hotmail.com) Daniel Hechler (dhechler@hotmail.com) Karen Rosenberger (Karen.rosenberger@charite.de) Doreen Luedecke (Doreen.Luedecke@charite.de) Eva M. Peters (Eva.peters@charite.de) Robert Nitsch (robert.nitsch@uni-mainz.de) Sven Hendrix (Sven.hendrix@uhasselt.be) ISSN 1742-2094 Article type Research Submission date 28 October 2011 Acceptance date 26 December 2011 Publication date 26 December 2011 Article URL http://www.jneuroinflammation.com/content/8/1/183 This peer-reviewed article was published immediately upon acceptance. It can be downloaded, printed and distributed freely for any purposes (see copyright notice below). Articles in JNI are listed in PubMed and archived at PubMed Central. For information about publishing your research in JNI or any BioMed Central journal, go to http://www.jneuroinflammation.com/authors/instructions/ For information about other BioMed Central publications go to http://www.biomedcentral.com/ Journal of Neuroinflammation © 2011 Boato et al. ; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro Francesco Boato 1, 2 *, Daniel Hechler 3, *, Karen Rosenberger 3 , Doreen Lüdecke 3 , Eva M. Peters 4,5 , Robert Nitsch 6 and Sven Hendrix 1,# 1 Dept. of Functional Morphology & BIOMED Institute, Hasselt University, Belgium; 2 present address: Institut de la Vision, Université Pierre et Marie Curie, Paris, France ; 3 Institute of Cell Biology and Neurobiology, Center for Anatomy, Charité – Universitätsmedizin Berlin, Germany; 4 Psychoneuroimmunology, University- Medicine Charité, Charité Center 12 for Internal Medicine and Dermatology, D-10117 Berlin, Germany; 5 Department of Psychosomatic Medicine, Justus-Liebig-University, Gießen, Germany; 6 Institute for Microscopic Anatomy and Neurobiology, University Medicine Mainz, Johannes Gutenberg University Mainz, Germany * FB and DH contributed equally to this study # corresponding author: Hasselt University - Campus Diepenbeek Dept. of Morphology & BIOMED Institute Agoralaan Gebouw C BE 3590 DIEPENBEEK Belgium Tel: +32 (0)1126 9246 Fax: +32 (0)1126 9299 Email: sven.hendrix@uhasselt.be Abstract Pro-inflammatory cytokines such as interleukin-1 beta (IL-1β) are considered to exert detrimental effects during brain trauma and in neurodegenerative disorders. Consistently, it has been demonstrated that IL-1β suppresses neurotrophin-mediated neuronal cell survival rendering neurons vulnerable to degeneration. Since neurotrophins are also well known to strongly influence axonal plasticity, we investigated here whether IL-1β has a similar negative impact on neurite growth. We analyzed neurite density and length of organotypic brain and spinal cord slice cultures under the influence of the neurotrophins NGF, BDNF, NT-3 and NT-4. In brain slices, only NT-3 significantly promoted neurite density and length. Surprisingly, a similar increase of neurite growth was induced by IL-1β. Additionally, both factors increased the number of brain slices displaying maximal neurite growth. Furthermore, the co-administration of IL-1β and NT-3 significantly increased the number of brain slices displaying maximal neurite growth compared to single treatments. These data indicate that these two factors synergistically stimulate two distinct aspects of neurite outgrowth, namely neurite density and neurite length from acute organotypic brain slices. Keywords: interleukin 1 beta, IL-1β, neurotrophin 3, NT-3, NGF, spinal cord, brain slices, neurite growth, axon outgrowth, neuroplasticity. Introduction Interleukin-1 beta (IL-1β) is a member of the IL-1 family of cytokines which have potent pro-inflammatory properties. It is produced in the periphery mainly by monocytes and is a strong activator of the host immune response to both injury and infection [1, 2]. In the central nervous system (CNS) IL-1β is primarily produced by microglia and invading monocytes/macrophages, but other types of resident cells of the nervous system, including neurons and astrocytes, are also capable of its production [3]. It is generally believed that inflammatory processes stimulated by pro- inflammatory cytokines and particularly by IL-1β, are rather detrimental and can aggravate the primary damage caused by infection of the CNS. This has been suggested by various in vivo studies, in line with its enhanced expression in the brain after damage or in neurodegenerative diseases, including Alzheimer’s disease (AD). Consistently, IL-1 deficient mice display reduced neuronal loss and infarct volumes after ischemic brain damage [4] and direct application of the recombinant cytokine results in an enhanced infarct volume [5]. In traumatic brain injury, antibodies against IL-1β reduce the loss of hippocampal neurons [6]. Consistently, in a mouse model of AD, an inhibitor of pro-inflammatory cytokine production suppressed neuroinflammation leading to a restoration of hippocampal synaptic dysfunction markers [7]. In AD it has also been demonstrated that members of the IL-1 family are associated with an increased risk of contracting the disease [8]. The findings in various in vitro models suggest a rather elaborated mechanism. In culture, IL-1β demonstrated neurotoxic effects towards hippocampal neurons exposed to high concentrations (500 ng/ml) combined with long-term exposure (three days). However, no effect was observed in lower concentrations following short-term exposure (one day) [9]. In other in vitro models, IL-1β has even been seen to display beneficial effects towards neuronal survival in the CNS [10, 11]. This has also been observed in axonal growth in the peripheral nervous system both in vivo following sciatic nerve injury [12, 13] and in vitro in adult dorsal root ganglion (DRG) collagen gel explant cultures [14], but not in dissociated single DRG neuron cultures [15]. Previously, it has been demonstrated that IL-1β impairs neurotrophin-induced neuronal cell survival [16, 17]. It has long been hypothesized that cytokine effects on neurite growth may be mediated at least in part by modulating neurotrophin signalling accordingly [18]. In addition to their positive effect on cell death, the neurotrophins Nerve Growth Factor (NGF), Brain-derived Neurotrophic Factor (BDNF), Neurotrophin-3 (NT-3) and NT-4 have also a well documented impact on axon plasticity and regeneration [19, 20]. This is crucial in the context of CNS insult to provide re-innervation and thus consecutive functional recovery. Based on these observations we investigated whether IL-1β is also a modulator of neurotrophin- induced neurite outgrowth in the CNS in vitro, using organotypic brain and spinal cord slice cultures. The present study shows that surprisingly, IL-1β did not abrogate NT- 3-induced neurite outgrowth but conversely showed a significant synergistic effect. These data indicate that IL-1β differentially regulates the effect of NT-3 on neuronal survival and neurite extension. Material und Methods Animals and factors C57BL/6 wildtype mice and IL-1β-deficient mice [21] were housed in a conventional animal facility (Center for Anatomy, Charité-Universitätsmedizin, Berlin, Germany). All experiments were performed in accordance with German guidelines on the use of laboratory animals. Recombinant neurotrophins NGF, BDNF, NT-3 and NT-4 were used in a concentration of 500 ng/mL (all Tebu-Bio, Offenbach, Germany). Recombinant IL-1β (Tebu-Bio, Offenbach, Germany) was used in concentrations of 5, 50 and 500 ng/mL. Acute organotypic brain slice culture The entorhinal slice cultures were prepared from mouse brains at postnatal day 2 as previously described [22-25]. In brief, after decapitation, the entorhinal cortex was dissected in ice-cold preparation medium, containing MEM with L-Glutamine (2mM) and Trisbase (8 mM). Transverse slices 350 µm thick were cut using a tissue chopper (Bachhofer, Reutlingen, Germany). Collagen was prepared as previously described [26]. Each entorhinal slice was embedded in a drop of collagen matrix on glass slides. The recombinant factors (neurotrophins and IL-1β) were mixed into the sterile cultivation medium containing MEM, 25% HBSS, 25% heat-inactivated normal horse serum, 4 mM L-glutamine, 4 µg/ml insulin (all from Gibco, Karlsruhe, Germany), 2.4 mg/ml glucose (Braun, Melsungen, Germany), 0.1 mg/ml streptomycin, 100 U/ml penicillin, and 800 ηg/ml vitamin C (all Sigma-Aldrich, Taufkirchen, Germany). The collagen co-cultures were incubated at 37°C in a humidified atmosphere with 5% CO 2 . After 48h in vitro, the collagen slices were analyzed microscopically (Olympus IX70, Hamburg, Germany). Neurotrophin concentrations were chosen after extensive pilot experiments based on studies by the Kapfhammer group on age-dependent regeneration of entorhinal fibers in mouse slice cultures [19], which showed that substantially higher concentrations are needed for brain slices compared to primary cell cultures. Measurement of axonal density and length of organotypic brain slice cultures To evaluate the axon outgrowth from entorhinal cortex explants, we improved a pragmatic, reliable and reproducible method, with which the axonal density and length was evaluated after two days in culture [23, 27]. Two independent blinded investigators evaluated neurite density on a scale from 0 (no axons) to 3 (multiple axons), at a total magnification of 200, using a 20x Olympus LCPLANFL objective (Olympus IX70, Hamburg, Germany). Axonal length was quantified at a total magnification of 100, using a 10x Olympus LCPLANFL objective and a widefield eyepiece with a grid of 100 x 100 µm (Olympus WH 10X2-H, Hamburg, Germany) and by measuring the length of a minimum of 10 axons growing in the same direction and reaching the same length: grade 0 (0 - 200 µm), 1 (200 - 400 µm), 2 (400 - 800 µm) and 3 (> 800 µm). Slices with a score equal 3 in length or density, where considered as having “maximum growth” and were then used for further analysis. For combined “maximum density and length” analysis, only the slices which reached the maximum score in both parameters were selected. All experiments were repeated at least three times. Acute organotypic transverse spinal cord slice cultures Transverse spinal cord cultures were prepared from mice at embryonic stage 13 (E13). After preparation out of the amniotic sac, embryos were decapitated and skin and organs were removed to isolate the spinal column, it was immediately transferred into ice cold HBSS medium. After dissection of the spinal cord, the remaining dorsal root ganglia (DRG) were removed and lumbar and cervical spinal sections dismissed. The thoracic segment was cut with a tissue chopper into 350 µm slices. These slices were divided along the sulcus medianus into two halves and each placed into a drop of collagen (as described above) with the cut surface of the sulcus medianus showing upwards. After polymerization of the collagen, 500µl of medium with or without factors were added to the slices. The transverse spinal cord slices were incubated at 37°C in a humidified atmosphere with 5% CO 2 . After 48h in vitro, the collagen slices were analyzed microscopically (Olympus IX70, Hamburg, Germany). Measurement of axonal outgrowth from transverse spinal cord slices Axonal outgrowth of the transverse spinal cord slices was evaluated as described previously for organotypic dorsal root ganglia cultures [28]. Slices were photographed in PBS with two fixed exposure times to visualize the neurite area and the slice, respectively. The ratio between these two areas was calculated and matched between slices with or without factor. All experiments were repeated at least three times. Statistical analysis The results are expressed as mean ± SEM. The values from the experimental cultures were compared to control cultures prepared in the same experiment (double treatment with NT-3 and IL-1β were additionally compared to single treatments). Subsequently, the data of each group were pooled for statistical analysis. After confirming that significant differences existed between the various groups by performing a Kruskal-Wallis Test, p-values were determined, using a Mann-Whitney- U test. A Chi 2 -test was used to test if the frequency of maximal neurite growth was significantly different between the groups. Results Previously, IL-1β has been described as a negative modulator of neurotrophin- induced neuronal survival [16, 17]. Therefore, we investigated whether IL-1β has a similar negative impact on NT-3-induced neurite growth from organotypic brain slices and transverse spinal cord slices. As a first step we investigated the effects of different neurotrophins on neurite growth in a classical model of organotypic brain slice cultures. Organotypic brain slices were embedded in a three-dimensional collagen matrix in the presence of 500 ng/mL NGF, BDNF, NT-3 or NT-4 or solvent. These concentrations were chosen after extensive pilot studies based on the landmark studies by the Kapfhammer group on regeneration of entorhinal fibers in murine slice cultures [19]. Neurite density and length was microscopically analyzed (Fig. 1). Compared to control brain slices, neurite density was significantly increased by about 20 % after cultivating with NT-3. It is important to note that an increase of 20% is close to the maximum increase of axon outgrowth which can be induced in brain slices with our method of analysis. Such an increase is not seen after administration of the other neurotrophins (Fig. 1A). Similarly, NT-3 also significantly increased the length of the cortical neurites when compared to untreated controls while the other neurotrophins had no effect on neurite length (Fig. 1B). Thus, only recombinant NT-3 (but not NGF, BDNF or NT-4) is capable of stimulating neurite outgrowth as well as neurite length from entorhinal cortical neurons (Fig. 1E, F). A Chi 2 test also revealed a significant increase in the number of slices reaching maximal neurite density and length in the presence of NT- 3, compared to untreated controls (Fig. 1C, D). Since the effect of the inflammation-associated cytokine IL-1β on repair mechanisms in the CNS is controversial, we analyzed as a second step IL-1β effects on neurite [...]... ischemic-reperfusion injury in rabbits [68] Since IL-1β had the capacity to stimulate neurite growth in brain slices, we tested if the same effect could be achieved in a de novo organotypic spinal cord slice model Surprisingly neither the single administration of IL-1β or NT-3, nor the combined administration of both factors had an influence on the measured neurite growth from the spinal cord slices These findings... by combined application of NT-3 and IL- 1beta A + B: Neurite outgrowth (neurite density A and neurite length B) was not influenced in the absence of endogenous IL-1β in IL-1β-deficient mice Heterozygous IL-1βdeficient and wildtype mice served as controls n: 50 slices C + D: The combined administration of NT-3 and IL-1β shows only a slight increase in neurite density and length, if compared to single... spinal cord astrocytes, stimulating their production and release of fibroblast growth factor-2, to increase motor neuron survival Exp Neurol 2002, 173(1):46-62 Mrak RE, Griffin WS: Interleukin-1, neuroinflammation, and Alzheimer's disease Neurobiology of aging 2001, 22(6):903-908 Pineau I, Lacroix S: Proinflammatory cytokine synthesis in the injured mouse spinal cord: multiphasic expression pattern and. .. IL-1β in this context include the induction of IL-6, tumor necrosis factor (TNF)-α and nitric oxide [53] and increased proliferation of macrophages [54] and astrocytes [55-57] in vitro and in vivo Both IL-6 and TNF-α are associated with stimulating properties of neurite growth It was demonstrated that TNF-α can support glia-dependent neurite growth in organotypic mesencephalic brain slices [58] and is... Alzheimer's disease with an interleukin-1alpha gene polymorphism Ann Neurol 2000, 47(3):361-365 Araujo DM, Cotman CW: Differential effects of interleukin-1 beta and interleukin-2 on glia and hippocampal neurons in culture Int J Dev Neurosci 1995, 13(3-4):201-212 Carlson NG, Wieggel WA, Chen J, Bacchi A, Rogers SW, Gahring LC: Inflammatory cytokines IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha impart neuroprotection... LB: Interleukin-1 stimulation of astroglial proliferation after brain injury Science 1985, 228(4698):497-499 Giulian D, Woodward J, Young DG, Krebs JF, Lachman LB: Interleukin-1 injected into mammalian brain stimulates astrogliosis and neovascularization J Neurosci 1988, 8(7):2485-2490 Giulian D, Young DG, Woodward J, Brown DC, Lachman LB: Interleukin-1 is an astroglial growth factor in the developing... a key factor in the hypothermia induced neurite outgrowth, also as a recombinant factor [24] The neuropoietic cytokine IL-6 is known to be a potent stimulating factor of neurite growth and regeneration in organotypic hippocampal slices [59] as well as in dorsal root ganglion cells [28] Furthermore, IL-1β is capable of activating the production of growth factors in CNSderived cells It induces NGF [60-62],... brain slices Surprisingly, only recombinant NT3 (but not NGF, BDNF or NT-4) was able to stimulate neurite outgrowth as well as neurite length from organotypic brain slices, also increasing the number of slices displaying maximal outgrowth This is in contrast to several single cell studies in which neurotrophins are highly efficient in promoting axonal growth [45-47] However, brain slices should be... LB: Interleukin 1 of the central nervous system is produced by ameboid microglia J Exp Med 1986, 164(2):594-604 Shaftel SS, Griffin WS, O'Banion MK: The role of interleukin-1 in neuroinflammation and Alzheimer disease: an evolving perspective J Neuroinflammation 2008, 5:7 Licastro F, Pedrini S, Caputo L, Annoni G, Davis LJ, Ferri C, Casadei V, Grimaldi LM: Increased plasma levels of interleukin-1, interleukin-6... T, Bender A, Nain M, Gemsa D: Role of macrophage cytokines in influenza A virus infections Immunobiology 1993, 189(3-4):340-355 Hildebrand F, Pape HC, Krettek C: [The importance of cytokines in the posttraumatic inflammatory reaction] Unfallchirurg 2005, 108(10):793-794, 796-803 Bauer J, Berkenbosch F, Van Dam AM, Dijkstra CD: Demonstration of interleukin-1 beta in Lewis rat brain during experimental . use, distribution, and reproduction in any medium, provided the original work is properly cited. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro Francesco. Fully formatted PDF and full text (HTML) versions will be made available soon. Interleukin-1 beta and neurotrophin-3 synergistically promote neurite growth in vitro Journal of Neuroinflammation 2011,. slices. Keywords: interleukin 1 beta, IL-1β, neurotrophin 3, NT-3, NGF, spinal cord, brain slices, neurite growth, axon outgrowth, neuroplasticity. Introduction Interleukin-1 beta (IL-1β) is